Live births from urine derived cells.

Here we report urine-derived cell (UDC) culture and subsequent use for cloning which resulted in the successful development of cloned canine pups, which have remained healthy into adulthood. Bovine UDCs were used in vitro to establish comparative differences between cell sources. UDCs were chosen as a readily available and noninvasive source for obtaining cells. We analyzed the viability of cells stored in urine over time and could consistently culture cells which had remained in urine for 48hrs. Cells were shown to be viable and capable of being transfected with plasmids. Although primarily of epithelial origin, cells were found from multiple lineages, indicating that they enter the urine from more than one source. Held in urine, at 4°C, the majority of cells maintained their membrane integrity for several days. When compared to in vitro fertilization (IVF) derived embryos or those from traditional SCNT, UDC derived embryos did not differ in total cell number or in the number of DNA breaks, measured by TUNEL stain. These results indicate that viable cells can be obtained from multiple species' urine, capable of being used to produce live offspring at a comparable rate to other cell sources, evidenced by a 25% pregnancy rate and 2 live births with no losses in the canine UDC cloning trial. This represents a noninvasive means to recover the breeding capacity of genetically important or infertile animals. Obtaining cells in this way may provide source material for human and animal studies where cells are utilized.

Blastocyst formation, embryo transfer and breed comparison in the first reported large scale cloning of camels.

Cloning, through somatic cell nuclear transfer (SCNT), has the potential for a large expansion of genetically favorable traits in a population in a relatively short term. In the present study we aimed to produce multiple cloned camels from racing, show and dairy exemplars. We compared several parameters including oocyte source, donor cell and breed differences, transfer methods, embryo formation and pregnancy rates and maintenance following SCNT. We successfully achieved 47 pregnancies, 28 births and 19 cloned offspring who are at present healthy and have developed normally. Here we report cloned camels from surgical embryo transfer and correlate blastocyst formation rates with the ability to achieve pregnancies. We found no difference in the parameters affecting production of clones by camel breed, and show clear differences on oocyte source in cloning outcomes. Taken together we demonstrate that large scale cloning of camels is possible and that further improvements can be achieved.

Establishment and characterization of embryonic stem-like cells from porcine somatic cell nuclear transfer blastocysts.

This study was aimed to establish embryonic stem (ES)-like cells from blastocysts derived from somatic cell nuclear transfer (SCNT) in pig. Somatic cells isolated from both day-30 fetus and neonatal cloned piglet were used for donor cells. A total of 60 blastocysts (46 and 14 derived from fetal and neonatal fibroblast donor cells, respectively) were seeded onto a mitotically inactive mouse embryonic fibroblast (MEF) monolayer and two ES-like cell lines, one from each donor cell type, were established. They remained undifferentiated over more than 52 (fetal fibroblast-derived) and 48 (neonatal fibroblast-derived) passages, while retaining alkaline phosphatase activity and reactivity with ES specific markers Oct-4, stage-specific embryonic antigen-1 (SSEA-1), SSEA-4, TRA-1-60 and TRA-1-81. These ES-like cells maintained normal diploid karyotype throughout subculture and successfully differentiated into embryoid bodies that expressed three germ layer-specific genes (ectoderm: beta-III tubulin; endoderm: amylase; and mesoderm: enolase) after culture in leukemia inhibitory factor-free medium. Microsatellite analysis confirmed that they were genetically identical to its donor cells. Combined with gene targeting, our results may contribute to developing an efficient method for producing transgenic pigs for various purposes.

Demetylation of the sex-determining region Y gene promoter and incidence of disorder of sex development in cloned dog males.

Canine cloning is occasionally accompanied by abnormal sexual development. Some male donor cells produce cloned pups with female external genitalia and complete male gonadal dysgenesis, which is classified as an XY disorder of sex development (XY DSD). In this study, we examine the potential of 5-aza-2'-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, to reduce the phenotypic abnormality XY DSD in somatic cell nuclear transfer (SCNT)- derived pups. We used a 9-year-old normal male German Shepherd dog as a cell donor. Donor cells were treated with 10 nM 5-aza-dC for 4 days before being used for SCNT. At the same stage of cell development, significantly lower levels of DNA methylation of the sex-determining region Y (SRY) promoter was observed in the treated donor cells compared to that in the untreated cells (95.2% versus 53.3% on day 4 for the control and treated groups, respectively). No significant differences were observed in the control or treatment groups concerning fusion rate, pregnancy rate (30 days or entire period), the number of pups, or the incidence of XY DSD. However, more XY DSD dogs were observed in the control group (31.25%) than in the treatment group (14.29%). Hypermethylation of the SRY promoter was observed in the XY DSD cloned pups in both the treatment (84.8%) and control groups (91.1 ± 1.4%) compared to the methylation level in the phenotypically normal male pups of the treatment (23.2 ± 20.9%) and control groups (39.1 ± 20.1%). These results suggest that 5-aza-dC treatment of donor cells can reduce the methylation level of the SRY promoter in donor cells, and thus, 5-aza-dC is advantageous for reducing the incidence of XY DSD in canine cloning.

Perforation predicts poor prognosis in patients with primary intestinal diffuse large B-cell lymphoma.

AIMS: To elucidate the clinicopathological features and prognostic factors of primary intestinal diffuse large B-cell lymphoma (PI-DLBL). METHODS AND RESULTS: Archival tissues from 30 tumours were used for tissue microarray construction, immunohistochemistry and interphase fluorescence in situ hybridization for chromosomal translocation. The M:F ratio was 1.7:1, with a median age of 60 years. The ileum and ileocaecum were most frequently involved (40% each). Fourteen (47%) were at stage I(E) disease, 15 (50%) at stage II(E). Five (17%) tumours were perforated at presentation. The tumours expressed Bcl-6 (73%), MUM1 (70%), Bcl-2 (67%) and CD10 (23%). Nine (30%) were classified as germinal centre B-cell (GCB) phenotype and 21 non-GCB. Eight of 30 (27%), 7/30 (23%) and 2/29 (7%) cases were positive for rearrangements involving IGH, BCL6, and C-MYC loci, respectively, whereas all cases were negative for BCL2 and CCND1 translocation. Perforation was a poor prognostic indicator, with a hazard ratio of tumour-related death at 8.75 (P = 0.001). The differentiation antigens, GCB versus non-GCB phenotype, or lymphoma-associated translocations were of no prognostic significance. CONCLUSIONS: We found a higher rate of perforation and lower frequency of GCB phenotype in PI-DLBL in Taiwan compared with other geographical areas; perforation is a poor prognostic indicator.

Effects of culture conditions and nuclear transfer protocols on blastocyst formation and mRNA expression in pre-implantation porcine embryos.

This study investigated the effects of culture conditions and somatic cell nuclear transfer (SCNT) protocols on in vitro development of porcine SCNT embryos and on expression patterns of genes involved in stress (heat shock protein 70.2, HSP70.2), trophoblastic function (integrin beta1, ITGB1), metabolism (phosphoglycerate kinase 1, PGK1), apoptosis (BAX), and imprinted gene (insulin-like growth factor 2 receptor, IGF2R). In Experiment 1, supplementing modified North Carolina State University (mNCSU) medium with 10% FBS at Day 4 of culture increased SCNT blastocyst formation (22.9 vs. 10.7%, P<0.05), number of inner cell mass cells (13.3+/-4.3 vs. 7.6+/-2.2, P<0.05), and total cells (57.9+/-19.5 vs. 36.3+/-8.2, P<0.05) in cloned blastocysts. In Experiment 2, using culture medium with 10% FBS, 1.0mM calcium in fusion/activation medium (1.0C), and 7.5mug/mL cytochalasin B treatment (0.1C&CB) yielded higher rates (P<0.05) of blastocysts (33.6 and 33.3%, respectively) relative to the control (0.1mM calcium fusion medium, 0.1C; 18.3%). Total cell numbers of blastocysts were increased (P<0.05) in 1.0C (77.4+/-28.9) compared to the control (58.5+/-22.6). In vitro-derived blastocysts had higher expression levels of BAX and lower levels of HSP70.2, IGF2R compared to their in vivo-derived counterparts. Supplementing culture medium with 10% FBS increased relative abundances of BAX mRNA in SCNT blastocysts relative to in vivo-derived blastocysts. The transcript level of ITGB1 in blastocyst from 0.1C&CB was lower than in vivo blastocysts. In conclusion, different culture conditions or SCNT protocols affected in vitro development of SCNT embryos and altered several important genes (BAX, HSP70.2, IGTB1, and IGF2R) compared to conventional in vivo-derived blastocysts.

Effect of Adding Cerium on Microstructure and Morphology of Ce-Based Inclusions Formed in Low-Carbon Steel.

Intra-granular Acicular Ferrite (IAF), as one of the most well-known desirable microstructure of ferrite with a chaotic crystallographic orientation, can not only refine the microstructure and retard the propagation of cleavage crack but also provide excellent combination of strength and toughness in steel. The effect of adding cerium on microstructure and controlling proper cerium-based inclusions in order to improve properties in low-carbon commercial steel (SS400) were investigated. The type of inclusions can be controlled by changing S/O ratio and Ce content. Without Ce modification, MnS is a dominate inclusion. After adding Ce, the stable inclusion phases change from AlCeO3 to Ce2O2S. The optimum amount of cerium, 0.0235 wt.%, lead in proper grain refinement and formation of cerium oxide, oxy-sulfide and sulfide inclusions. Having a high amount of cerium results in increasing the number of inclusions significantly as a result it cannot be effective enough and the inclusions will act like barriers for others. It is found that the inclusions with a size of about 4∼7 µm can serve as heterogeneous nucleation sites for AF formation. Thermodynamic calculations have been applied to predict the inclusion formation in this molten steel as well, which show a good agreement with experimental one.

Effects of combined expression of human complement regulatory proteins and H-transferase on the inhibition of complement-mediated cytolysis in porcine embryonic fibroblasts.

The expression of human complement regulatory proteins (CRP) and H-transferase (HT) in porcine cells is one of the strategies for suppression of hyperacute rejection (HAR) of xenotransplants in human recipients. In this study, we investigated the inhibitory effect of combined expression of human complement regulators and HT on human serum-mediated cytolysis in porcine embryonic fibroblasts. For the combinated expression of human CRPs in transformed pig cells, cDNAs of human DAF, MCP, and CD59 were cloned into the same insertional plasmid under the control of pCMV IE and LTR. The double combination of CRPs, hDAF-hMCP, and hMCP-hCD59 survived over 50% in the presence of 50% human serum, compared to the control. Moreover, the cell viability was increased more than 65% and 80% in the combination of human DAF-CD59 and DAF-MCP-CD59, respectively. In addition, the combination of HT gene to hDAF-hCD59 vector increased the viability close to 80%, similar to the triple combination of CRPs. These observations suggest that the combined expression of human CRPs and HT in the same insertional vector may be more effective in protecting porcine cells from human complement-mediated cytolysis.

Direct comparisons of peripheral T-cell lymphoma with diffuse B-cell lymphoma of comparable histological grades--should peripheral T-cell lymphoma be considered separately?

Peripheral T-cell lymphoma (PTCL) forms a morphologically heterogeneous group of non-Hodgkin's lymphomas (NHL) with distinct immunophenotypes of mature T cells. Progress has been slow in defining specific clinicopathological entities to this particular group of NHL. In order to elucidate the specific characteristics of PTCL, a direct comparison of PTCL with a group of diffuse B-cell lymphomas (DBCL) was performed. Between June 1983 and December 1987, we studied 114 adults with NHL, using a battery of immunophenotyping markers. Adult T-cell leukemia/lymphoma, lymphoblastic lymphoma, mycosis fungoides/Sézary syndrome, follicular lymphoma, well-differentiated lymphocytic lymphoma, and true histiocytic lymphoma were excluded from this study since these are distinct clinicopathologic entities with well-recognized immunophenotypes. Of the remaining 75 patients, 70 who had adequate clinical information were analyzed, and of these, 34 were PTCL and 36 were DBCL. Classified according to the National Cancer Institute (NCI) Working Formulation (WF), 68% of PTCL and 31% of DBCL were high-grade lymphomas. Clinical and laboratory features were similar, except PTCL had a characteristic skin involvement and tended to present in more advanced stages with more constitutional symptoms. Induction chemotherapy was homogeneous in both groups, and complete remission rates were 62% for PTCL and 67% for DBCL. Patients with DBCL had a better overall survival than patients with PTCL, but the survival benefit disappeared after patients were stratified according to intermediate- or high-grade lymphoma. A subgroup of PTCL patients who had received less intensive induction chemotherapy was found to have a very unfavorable outcome. We conclude that (1) PTCL follows the general grading concept proposed in WF classification; (2) within a given intermediate or high grade, PTCL and DBCL respond comparably to treatment; (3) the intensity of induction chemotherapy has a crucial impact on the outcome of PTCL patients; and (4) with a few exceptions, the clinical and laboratory features of PTCL and DBCL are comparable.

The effect of PDE5 inhibition on the erectile function in streptozotocin-induced diabetic rats.

The aim of this study was to assess the effect of phosphodiesterase 5 inhibitor, DA-8159, on erectile function throughout the quantitative analysis of vascular endothelial cell, smooth muscle (SM), TGF-beta1 expression in rat corpus cavernosum and measurement of intracavernous pressure (ICP) in diabetic rats. DA-8159 (0, 5, 10, 20 mg/kg) was administered orally once a day to diabetic rats. After 8 weeks, immunohistochemistry and computerized image analysis were performed to quantify the percent area within the Corpora Cavernosa occupied by the endothelial cells, SM cells and fibrotic tissues. ICP/mean arterial pressure (MAP) was also measured by electrostimulation of the cavernous nerve. Diabetic rats showed a significant decrease in the SM and endothelial cell content, and an increase in the TGF-beta1 expression level within the cavernosa areas compared to the normal rats. The mean cavernous SM, endothelial cell content and TGF-beta1 expression level were 9.7+/-0.7, 4.5+/-0.7 and 17.9+/-2.1%, respectively. DA-8159 prevented reduction of SM (12.3+/-0.4% (5 mg/kg), 13.8+/-0.4% (20 mg/kg)) and endothelial cell content (5.6+/-0.5% (5 mg/kg), 6.3+/-0.6% (20 mg/kg)). Immunoreactivity of TGF-beta1 and intracorporal fibrosis were also significantly lower in DA-8159-treated groups (11.8+/-1.2% (5 mg/kg), 9.5+/-1.1% (20 mg/kg)). Electrostimulation of the cavernous nerve induced significant increase in maximum ICP (62.2+/-13.6 mmHg in 10 mg/kg vs 37.5+/-17.5 mmHg in diabetic group) and area under the curve of the ratio of ICP/MAP (8891.09+/-1957 in 10 mg/kg vs 6315.87+/-2272 in diabetic group). These results suggest that subchronic treatment of DA-8159 can prevent the development of erectile dysfunction (ED), and provides a rationale for the use of DA-8159 as treatment of diabetic ED.

High oxygen tension during in vitro oocyte maturation improves in vitro development of porcine oocytes after fertilization.

This study was conducted to evaluate the effect of oxygen tension during IVM and/or IVC on developmental competence of porcine follicular oocytes. Prospective, randomized experiments were designed, and oocytes were matured, inseminated and cultured in vitro in the designated condition. In experiment 1, either high (20%) or low (7%) oxygen tension was used for IVM. The high oxygen significantly improved blastocyst formation (23% versus 13%; P<0.01) after IVF than the low oxygen. Such treatment, however, did not significantly (P>0.05) improve the rates of nuclear maturation (89% in each treatment), sperm penetration (62-72%), monospermic fertilization (56-67%), pronuclear formation (90-96%), cleavage (49-53%) and blastocyst cell number (31-32 cells). In experiment 2, the combined effect of oxygen tension during IVM and IVC of embryos was evaluated by a 2 x 2 factorial arrangement. Again, the high oxygen tension during IVM supported blastocyst formation more efficiently (P<0.01) than the low oxygen, and this was independent of oxygen tension during IVC (26-28% versus 15-16%). In oocytes matured under the high oxygen, a tendency to increase blastomere number (P=0.0630) was found, when the low oxygen was used for IVC after insemination (39-45 cells/blastocyst). In conclusion, the use of high oxygen tension (20% maintained by exposure to 5% CO2 in air) for IVM of porcine oocytes promoted blastocyst formation in vitro.

Omental infarction in children: imaging features with pathological correlation.

INTRODUCTION: Omental infarction is a rare occurrence in the paediatric population. It often presents as an acute abdomen that can mimic acute appendicitis and cholecystitis. METHODS: Six cases of omental infarction in children, proven on histopathology, were retrospectively reviewed for their clinical presentation and imaging findings on ultrasonography and computed tomography. RESULTS: These cases revealed clinical and imaging findings on computed tomography that were suggestive and helpful in the pre-operative diagnosis of omental infarction. Findings on ultrasonography were less specific. Histopathological specimens revealed findings of vasculitis in all cases. CONCLUSION: There are clinical and imaging features that will help in the pre-operative diagnosis of this uncommon condition. We also postulate vasculitis as a possible underlying pathology for omental infarction.

Fetus-in-fetu in the pelvis: report of a case and literature review.

INTRODUCTION: Fetus-in-fetu is an extremely rare condition in which a malformed fetus is found in the body of its twin. To our knowledge, fewer than 100 cases have been reported. Wide variations of presentation have been described, although its embryo-pathogenesis and differentiation from a teratoma have not been well established. CLINICAL PICTURE: We describe a male neonate with a fetoid-like mass in his pelvis associated with bilateral undescended testes. The mass was detected on prenatal ultrasound scans. The diagnosis of fetus-in-fetu was considered prenatally and confirmed on a computed tomography scan after birth. OUTCOME: The mass was successfully excised. Histological examination, accompanied by a review of the literature, confirmed that the mass had features consistent with a fetus-in-fetu. CONCLUSIONS: Although an extremely rare clinical entity, fetus-in-fetu can be diagnosed prior to surgery with current imaging modalities. When it arises in the retroperitoneum of a male infant, it can hinder the descent of the testes. Complete excision is curative.

Neotropical Physoderinae revisited, with description of a new, sexually dimorphic species of Leptophysoderes Weirauch (Hemiptera: Reduviidae).

The small reduviid subfamily Physoderinae has the greatest species diversity in the Oriental region and Madagascar. Only the two monotypic genera Cryptophysoderes Wygodzinsky and Maldonado and Leptophysoderes Weirauch are currently known from the Neotropical region. We here describe and document a new, sexually dimorphic species of Physoderinae, Leptophysoderes sarapiqui sp. nov. from Costa Rica. The generic diagnosis of Leptophysoderes is modified to accommodate the new species. Females and immatures of Leptophysoderes are documented for the first time.

Idiopathic retractile (sclerosing) mesenteritis and its differential diagnosis.

We report a case of retractile mesenteritis presenting as an abdominal mass with incomplete small-bowel obstruction. Histological features included fat necrosis, fibrosis, elastosis, dystrophic calcification, and chronic inflammation. Lymphatic obstruction resulted in the accumulation of lipid-laden macrophages in the ileal mucosa. Ultrastructurally, myofibroblasts were the principal cells present. The differential diagnosis of retractile mesenteritis is discussed with particular attention to myofibroblastic disorders such as inflammatory pseudotumors, desmoids, retroperitoneal fibrosis, and other uncommon conditions that appear to be morphologically or clinically distinguishable although the etiology and pathogenesis are obscure.

Gated myocardial perfusion tomography for the assessment of left ventricular function and volumes: comparison with echocardiography.

UNLABELLED: The purpose of this study was to evaluate left ventricular volumes and function by gated SPECT using different tracers and protocols in comparison with quantitative echocardiography. Gated myocardial perfusion scintigraphy permits simultaneous assessment of left ventricular perfusion, function and volumes. Information is scanty regarding the accuracy of absolute left ventricular volumes measurements by this technique. METHODS: We performed gated SPECT and echocardiography within 15 d of each other in 109 consecutive patients (53 men, 56 women; mean age 63 +/- 14 y). Gated tomographic data, including left ventricular volumes and ejection fraction, were processed using an automatic algorithm, whereas echocardiography used standard techniques. RESULTS: The correlations between gated tomography and echocardiography with respect to end-diastolic volume, end-systolic volume and left ventricular ejection fraction were good to excellent (all P < 0.001, r values > or = 0.68), regardless of the use of poststress or rest/redistribution images, 201Tl or 99mTc tracers. End-systolic volume was similar with gated tomography and echocardiography (P = ns), but end-diastolic volume and left ventricular ejection fraction were significantly higher with echocardiography (P < or = 0.05). CONCLUSION: Quantitative gated tomography, using either 201Tl or 99mTc tracers, has a good correlation with echocardiography for the assessment of left ventricular volumes and ejection fraction. These results support the clinical use of this new technique.

Prediction of chemotherapy response in human leukemia using in vitro chemosensitivity test.

We performed the dye exclusion assay (DEA) and the MTT dye reduction assay to determine the drug sensitivity of acute leukemia using short-term microplate cultures. The in vitro results were compared with the clinical response of 31 patients with acute leukemia treated by combination chemotherapy. The true-positive rates of the DEA and MTT assays were 86.7 and 91.3%, respectively; the true-negative rates were 33.3 and 77.8%, respectively; and the predictive accuracy was 62.5 and 87.5%, respectively. The DEA and MTT assays gave comparable results in drug sensitivity testing of leukemic blast cells. Our data suggest that MTT assay is the more suitable for assessing chemosensitivity in acute leukemia.

Diffuse-variant tenosynovial giant cell tumor: a rare and aggressive lesion.

The diffuse-variant tenosynovial giant cell tumor is rare. Although it shares histologic features with the exclusively intra-articular pigmented villonodular synovitis and local tenosynovial giant cell tumor, its behavior differs dramatically, being locally very aggressive. We report a case of a diffuse-variant aggressive tenosynovial giant cell tumor that, although diploid by flow cytometry, demonstrated trisomy 7 and 5 as well as clonal rearrangements involving chromosomes 1, 3, and 15. These cytogenetic abnormalities may be markers for aggressive behavior and useful for directing treatment.

Signet-ring chondrosarcoma: a new morphologic entity.

A 65-year-old, otherwise healthy white man presenting with an asymptomatic anterior chest wall mass diagnosed from a routine preoperative chest x-ray is reported. A fine needle aspirate of the mass was initially interpreted as a metastatic adenocarcinoma with prominent "signet-ring" features, but ultrastructural study of the cell block later suggested a chondrosarcoma. The resected surgical specimen confirmed the diagnosis of a grade 2 chondrosarcoma, with most of the tumor cells containing a large, clear, single vacuole shown to be lipid. The positive immunostaining for vimentin and S-100 as well as the ultrastructural appearance confirmed the diagnosis of a chondrosarcoma. Flow cytometric DNA analysis of the tumor on two separate occasions documented a very large aneuploid cell population (50% to 60%) which, when interpreted with the histologic appearance, suggested an aggressive tumor. This case illustrates the first published example of a "signet-ring" chondrosarcoma.